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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 13, 2006 as doi:10.1096/fj.05-4341fje. |
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,1
,
* Department of Physiology,
Vascular Biology Center, and
Department of Surgery, Urology Section, Medical College of Georgia, Augusta, Georgia, USA;
Department of Urology, Johns Hopkins University, Baltimore, Maryland, USA; and
|| Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, USA
1Correspondence: Department of Urology, Johns Hopkins University, Baltimore, MD 21287, USA. E-mail: ljin8{at}jhmi.edu
SPECIFIC AIMS
Erectile dysfunction (ED) is estimated to affect 152 million men worldwide. The aim of this study is to identify new vasodilatory mechanisms involved in erectile function. We hypothesized that epoxyeicosatrienoic acids (EETs) produced by cytochrome P450 (CYP) epoxygenases function as endothelium-derived relaxing factors (EDRFs) in penis and are essential to achieve full erection.
PRINCIPAL FINDINGS
1. Detection and localization of CYP epoxygenases and NADPH CYP reductase in rat penile tissue
Protein expressions of CYP epoxygenases were examined using Western blot analysis. CYP epoxygenases are membrane-bound, heme-containing enzymes and require NADPH CYP reductase as an electron-transferring protein to complete the oxidation of the substrates. In rat corpora cavernosa, CYP2C9, CYP2C23, CYP2B1/2 and CYP2J2 protein expressions were observed using specific antibodies. Results showed that NADPH CYP reductase is also expressed in rat corpora. The experiments prove that the enzyme system required for the synthesis of vasodilatory EETs is present in the rat penis.
Next we examined whether CYP epoxygenases are located in cavernosal sinusoids. Cryosectioned penis tissue was stained with CYP2C9 antibody. Intensive staining of CYP2C9 was found on the luminal side of cavernosal space and cavernosal arterioles but was not seen in the negative control sections, which were incubated without CYP2C9 antibody or with normal rabbit IgG.
We determined whether CYP epoxygenases are localized in corporal endothelial cells using double immunolabeling of rat penis tissue for CYP2C9 and von Willebrand’s factor. The sinusoidal endothelium layer was labeled with von Willebrand’s factor, a widely used marker for endothelial cells. The location of CYP2C9 was detected with CYP2C9 antibody. When the images were superimposed it was apparent that CYP2C9 was colocalized with von Willebrand’s factor, suggesting that CYP2C9 is present in sinusoidal endothelial cells.
2. Reverse-phase HPLC analysis for metabolites of CYP epoxygenases isolated from corpora cavernosa
Although four EET regioisomers (5,6-, 8,9-, 11,12-, and 14,15-EET) have been found to be vasodilators in different microvascular beds, 11,12- and 14,15-EET are the most potent vasodilators. Epoxygenase activity was measured by reverse-phase HPLC using radio-labeled [1-14C] arachidonic acid (AA) (7 nmol) as substrate. Microsomes were made from rat penile tissue in order to concentrate epoxygenases because these enzymes are enriched in the endoplasmic reticulum. After incubation of corpus cavernosal microsomes with [1-14C]-AA for 30 min, the mixture was analyzed for EETs production and results were compared with authentic EET standards. The HPLC analysis results show that all four authentic EET regioisomers can be separated in this system. The 11,12-EET standard was detected between 16.5 and 17.5 min. The major product of CYP epoxygenases in corpus cavernosum appeared to be 11,12-EET with a retention time identical to that of authentic 11,12-EET; the other three regioisomers were not detectable. The conversion rate of AA to 11,12-EET is 1.5 pmol/mg protein/min.
3. Effect of EETs inhibition on erectile function in vivo
Next, we investigated the effect of inhibiting CYP epoxygenase pathway on erectile function in anesthetized rats by measuring intracavernosal pressure (ICP) and mean arterial blood pressure (MAP) in the presence or absence of an EETs inhibitor. 14,15-Epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) is a highly selective EETs inhibitor with a structure similar to 14,15-EET except for the absence of olefin bonds between carbons 8,9 and 11,12. 14,15-EEZE has been demonstrated to directly inhibit 5,6-, 8,9-, 11,12-, and 14,15-EET-mediated endothelium-dependent relaxation in coronary artery.
Injection of 5 µL of 10 mM 14,15-EEZE (150 nmol/kg body weight) into rat corpora had no effect on MAP, suggesting 14,15-EEZE does not have significant systemic effects at the dose used in these experiments. However, erectile responses (expressed as ICP/MAP) were markedly reduced after injection with this dose of 14,15-EEZE compared with ICP/MAP measured before injection in the same animal (0.45±0.02 vs. 0.64±0.01 before injection; n=5, P<0.01). Figure 1
A shows representative tracings of ICP and MAP recorded simultaneously upon electrical stimulation of the major pelvic ganglion (MPG) before and after injection of 14,15-EEZE. Administration of 5 µL vehicle (ethanol) did not significantly affect the ICP/MAP (0.65±0.03 vs. 0.67±0.03 before injection; n=5) as shown in Fig. 2B
with representative tracings of ICP and MAP. The data are summarized in Fig. 2C
and expressed as ICP/MAP measured after injection of 14,15-EEZE as percent of ICP/MAP measured before injection in the same animal. The inhibitory effect of 14,15-EEZE on the erectile response was dose dependent. When the dose of 14,15-EEZE was decreased to 75 nmol/kg, inhibition was reduced to 23% of ICP/MAP measured before injection.
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Repeated stimulation of the MPG had no significant effect on ICP/MAP, which is 0.66 ± 0.02, 0.65 ± 0.02 and 0.67 ± 0.02 for the 1st, 2nd, and 3rd stimulations before injection of drugs, respectively. After administration of 14,15-EEZE (150 nmol/kg body weight), ICP/MAP was significantly decreased upon the 1st stimulation of the MPG (0.45±0.02). ICP/MAP was almost normal compared with those before injection upon the 2nd and the 3rd stimulations (0.62±0.04 and 0.67±0.04 vs. 0.65±0.02 and 0.67±0.02 for ICP/MAP measured before injection). A similar result was observed in rats that received a lower dose of 14,15-EEZE (0.62±0.04, 0.67±0.03, and 0.69±0.02 for the 1st, 2nd, and 3rd stimulations after injection, respectively, vs. 0.69±0.05, 0.69±0.03, and 0.71±0.02 for ICP/MAP before injection). These data suggest that the inhibitory effect of 14,15-EEZE on erectile function is reversible.
CONCLUSIONS AND SIGNIFICANCE
To our knowledge, this is the first study providing direct evidence that EETs are produced in rat penis and function as EDRFs causing cavernosal smooth muscle relaxation. This CYP epoxygenase signaling pathway is an important vasorelaxation pathway required for normal erectile function.
It has been proposed that penile erection involves two distinct hemodynamic stages. The first stage depends on the release of NO from neuronal NO synthase (nNOS) containing nerve endings in the penis, causing the dilation of penile arterioles and expansion of the sinusoids so that a large amount of blood flows into the cavernosal sinuses. The second stage occurs when the expansion of sinuses and blood flow-induced shear stress stimulate the release of EDRFs from sinusoid endothelial cells, leading to achievement and maintenance of full erectile responses. Studies have shown that NO produced by endothelial NO synthase (eNOS) is a major vasodilator for the second stage of erection. Gene therapy with NOS isoforms has reversed some age-related decline in erectile responses in rats. There is also evidence suggesting that endothelium-derived NO may not be the only EDRF involved in the erectile response: 1) eNOS knockout mice have apparently normal erections even though they lose >40% of normal NOS activity, suggesting that possible vasodilatory mechanisms are up-regulated in the absence of eNOS activity; 2) gene therapy with eNOS fails to totally restore erectile responses in animal models of diabetes; and 3) clinical statistics shows that phosphodiesterase, (PDE 5) inhibitors have much lower success rates when administered to diabetic patients. In vitro studies demonstrate that PDE 5 inhibitors have no significant effects on acetylcholine-induced relaxation in human penile resistant vessels from diabetic patients but markedly increased relaxation in arteries from nondiabetic patients. It is reasonable to speculate that signaling pathways other than NO are required to facilitate and maintain full erection.
Studies have shown EETs are synthesized in various tissues by epoxygenases including members of CYP3A, CYP2B, CYP2C, and CYP2J subfamilies. We found that CYP2B1/2, CYP2C9, CYP2C23, and CYP2J2 epoxygenases were expressed in rat penile tissue. However, the relative importance of each of these enzymes in producing EETs in the rat penis has yet to be determined. The activity of CYP epoxygenases is regulated by hormonal or chemical stimulation as well as mechanical stimuli such as shear stress and stretch. Shear stress inducing NO-independent vasodilation as a mechanism has been found in coronary conduit and resistant vessels of canine, porcine, and human. The vasodilation is blocked by large-conductance calcium-dependent K+ channels (BKCa) channel blockers, high extracellular K+ concentration, and CYP epoxygenase inhibitors, suggesting EETs are released as endothelium-derived hyperpolarization factors in response to shear stress. Acute stimulation of stretch enhances CYP2C activity; chronic stretch increases CYP2C mRNA and protein synthesis in coronary endothelium. Our data show the presence of CYP epoxygenases and EETs metabolites in rat corporal tissue, providing fundamental evidence that the CYP epoxygenase signaling pathway may be involved in erection. A significant decrease in ICP/MAP after injection of a highly selective EET inhibitor, 14,15-EEZE, further demonstrates EETs are essential for complete erection. It is possible that the release of EETs in the penis is dependent on the blood flow-induced shear stress and the stretch of the endothelium layer after increased blood volume in the cavernous sinuses and penile resistance arterioles.
Studies have suggested that the NO and EETs signaling pathways are in an equilibrium state. NO has negative effects on EETs release through direct inhibition of CYP2C activity by binding to the heme moiety or by a decrease in CYP2C protein expression. Conversely, EETs positively regulate NOS protein expression through mitogen activated protein kinase and protein kinase C. In cases where NO production is impaired, increased release of EETs may become one of the compensatory mechanisms to maintain the microvascular tone. Flow-induced shear stress has been shown to result in arteriole smooth muscle relaxation in eNOS knockout mice or rats treated with a NOS inhibitor, which was abolished by CYP2C inhibitors but not a cyclooxygenase inhibitor. In patients with coronary artery disease (CAD), flow-induced vasodilation in atria and ventricle arterioles is mediated by EETs only, whereas NO is responsible for the flow-induced vasodilation in patients without CAD.
Based on our novel findings that vasodilation induced by CYP epoxygenase metabolites is required for normal erectile function, we propose the possible working mechanism of which EETs are involved in the regulation of penile erection (Fig. 2
). Sexual stimuli induce NO release from nerve endings in the penis, increasing blood flow in penile arterioles and sinuses. Blood flow-induced shear stress and expansion of cavernosal sinusoids stimulate the release of EETs and NO from endothelial cells in the penis, facilitating and maintaining the full erection. Reduced production of EETs and/or NO results in attenuated cavernosal smooth muscle relaxation and subsequently leads to ED.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4341fje;
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